This proposal presents a comprehensive account of an experimental plan aimed at exploring the mechanism of enzyme mediated phosphoryl group transfer. The long range goal of this project is to determine how catalysis of the transfer of a phosphoryl group from one metabolite to another in biological systems is effected. With the exception of the continued investigation of the mechanism of action of yeast inorganic pyrophosphatase that is planned, studies will focus on enzymes which catalyze the transfer of a phosphoryl group to and from carbon centers. The specific aims of this proposal are: (1) the purification, characterization and mechanistic investigation of the novel Tetrahymena pyriformis F.C bond forming enzyme. PEP phosphomutase which catalyzes the isomerization reaction: PEP forms and is formed from phosphonopyruvate. (2) the determination of the structure, mechanism of action and origin of the Bacillus cereus P.C bond cleaving enzyme, phosphonoacetaldehyde phosphohydrolase which catalyzes the reaction: phosphonoacetaldehyde gives acetaldehyde + Pi. (3) The elucidation of the biosynthetic pathway leading to the novel amino acid, phosphonotyrosine in Actinomadura and isolation and characterization of the enzyme catalyzing the key P- C bond forming step in the pathway. (4) The purification and mechanistic study of two P-C bond cleaving enzymes in T. pyriformis phosphonopyruvate phosphohydrolase (which catalyzes to conversion of phosphonopyruvate to pyruvate plus Pi) and phosphonoacetaldehyde phosphonohydrolase. (5) Determination of the mechanism of action of the yeast enzyme, inorganic pyrophosphatase which, with the assistance of Mg2+, catalyzes the conversion of inorganic pyrophosphate to orthophosphate.